Peptide derivative, and pharmaceutically acceptable salt thereof, process for producing the same, and use thereof

ABSTRACT

The present invention provides a peptide derivative of formula (I) or a pharmaceutically acceptable salt thereof 
                         
wherein X is —CH(OH)— or —CO—; R 1  is hydrogen or an amino protecting group; R 2  is hydroxyl or lower alkoxy; one of R 3  and R 4  is the side chain (R group) of lysine the amino group of which may be protected with a protecting group and the other of R 3  and R 4  is the side chain (R group) of arginine the guanidino group of which may be protected with a protecting group; and R 5  and R 6  may be the same or different and are hydrogen, lower alkyl or aralkyl. The present invention further provides a production process and use thereof.

TECHNICAL FIELD

The present invention relates to a novel peptide derivative andpharmaceutically acceptable salts thereof, and a production process anduse thereof.

BACKGROUND ART

Most periodontal diseases are considered to be a kind of infectiousdisease caused by indigenous microbes residing in the periodontal area.Among such indigenous microbes, a gram-negative anaerobic bacteriumcalled Porphyromonas gingivalis (hereinafter abbreviated as “P.gingivalis”) has been revealed to be the most important pathogenicbacterium that causes adult periodontitis and rapidly progressiveperiodontitis (J. Clin. Periodontol., 15, 85–93, 1988; ibid 316–323,1988; J. Dent. Res., 63, 441–451, 1984). In recent years, it has becomeknown that proteases produced by P. gingivalis decompose periodontaltissue components such as collagen and blood serum proteins involved inthe body's natural defense system and the proteases have revealed to beclosely related to the pathogenicity of P. gingivalis (Greiner D.,Mayrand D.: Biology of the Species Porphyromonas Gingivalis, Edited byShah H. N., Mayrrand D. and Genco R. J., pp. 227–243, CRC Press, BocaRaton, Ann Arbor, London, Tokyo, 1993).

Several kinds of proteolytic enzymes having trypsin-like proteaseactivity and produced by P. gingivalis are known. Among them,Lys-gingipain (hereinafter sometimes abbreviated as “KGP”) andArg-gingipain (hereinafter sometimes abbreviated as “RGP”) are theprincipal proteolytic enzymes. These enzymes are known to have potentdigestive activities to decompose high molecular weight kininogen andfibrinogen and are considered to be involved in bacterial attachment,the onset of periodontal disease and periodontal tissue destruction (J.Biol. Chem., 269, 406–411, 1994).

Conventionally, drugs for inhibiting the growth of bacteria are used toprevent and treat periodontal disease. Examples of such drugs includeantibiotics such as tetracycline and minocycline; natural products suchas chamomile tincture and rhatany tincture; cyclohexadine, tranexamicacid and the like. However, these drugs have safety problems or othervarious problems such as unpleasant smell. Japanese Unexamined PatentPublication No. 1993-97708 discloses periodontal therapeutic agentscomprising an ATPase inhibitor, a cysteine protease inhibitor or thelike as an active ingredient. Japanese Unexamined Patent PublicationsNos. 1999-139947 and 2000-191487 disclose a composition for use in theoral cavity comprising a matrix metalloprotease inhibitor as an activeingredient. However, the anti-periodontal disease effects of thetherapeutic agent and the composition are not satisfactory. Recentlyknown inhibitors for selectively inhibiting RGP are malabaricon Cdescribed in Japanese Unexamined Patent Publication No. 1999-335274 andan arginine derivative described in Japanese Unexamined PatentPublication No. 1999-228526. A lysine derivative described in TissueCulture Engineering, 27 (9), 343–347, 2001 is known as an inhibitorcapable of selectively inhibiting KGP.

However, these publications do not describe a compound capable ofinhibiting both enzymes, KGP and RGP.

DISCLOSURE OF THE INVENTION

An object of the invention is to provide a novel compound that haspotent inhibitory activity against both enzymes, KGP and RGP, thus beinguseful as a periodontal preventive or therapeutic agent, and provide aproduction process thereof.

Another object of the invention is to provide a novel inhibitor capableof inhibiting both KGP and RGP, a pharmaceutical preparation forperiodontal disease and a composition for use in the oral cavity.

A further object of the invention is to provide a novel method forpreventing or treating periodontal disease.

Other objects and features of the invention will become apparent fromthe following description.

To produce an effective preventive and therapeutic agent for periodontaldisease, the present inventors carried out intensive research focusingon the following points: P. gingivalis plays a significant role in theonset and progress of periodontal disease; and the proteolytic enzymesKGP and RGP both contribute to periodontal diseases caused by P.gingivalis. As a result, the inventors found a novel peptide derivativecapable of potently inhibiting the enzymatic activities of bothproteases, KGP and RGP. The inventors carried out further research basedon the above finding and accomplished the present invention.

The present invention provides the following novel peptide derivativeshaving inhibitory activity against both KGP and RGP, pharmaceuticallyacceptable salts thereof, and production processes and uses thereof.

1. A peptide derivative of formula (I) or a pharmaceutically acceptablesalt thereof

wherein X is —CH(OH)— or —CO—; R¹ is hydrogen or an amino protectinggroup; R² is hydroxyl or lower alkoxy; one of R³ and R⁴ is the sidechain (R group) of lysine the amino group of which may be protected witha protecting group and the other of R³ and R⁴ is the side chain (Rgroup) of arginine the guanidino group of which may be protected with aprotecting group; and R⁵ and R⁶ may be the same or different and arehydrogen, lower alkyl or aralkyl.

2. The peptide derivative of formula (I) or a pharmaceuticallyacceptable salt thereof according to item 1 wherein X is —CO—.

3. The peptide derivative of formula (I) or a pharmaceuticallyacceptable salt thereof according to item 2 wherein X is —CO—; R¹ ishydrogen or optionally substituted aralkyloxycarbonyl; R² is hydroxyl orlower alkoxy; one of R³ and R⁴ is the side chain (R group) of lysine andthe other of R³ and R⁴ is the side chain (R group) of arginine theguanidino group of which may be protected with a nitro group; and R⁵ andR⁶ may be the same or different and are hydrogen or aralkyl.

4. The peptide derivative of formula (I) or a pharmaceuticallyacceptable salt thereof according to item 3 wherein X is —CO—; R¹ isbenzyloxycarbonyl; R² is hydroxyl or t-butoxy; R³ is the side chain (Rgroup) of lysine; R⁴ is the side chain (R group) of arginine theguanidino group of which is protected with a nitro group; R⁵ ishydrogen; and R⁶ is phenethyl.

5. A process for preparing a peptide derivative of formula (I) or apharmaceutically acceptable salt thereof

wherein X is —CH(OH)— or —CO—; R¹ is hydrogen or an amino protectinggroup; R² is hydroxyl or lower alkoxy; one of R³ and R⁴ is the sidechain (R group) of lysine the amino group of which may be protected witha protecting group and the other of R³ and R⁴ is the side chain (Rgroup) of arginine the guanidino group of which may be protected with aprotecting group; and R⁵ and R⁶ may be the same or different and arehydrogen, lower alkyl or aralkyl,

the process comprising the following step (i):

-   (i) condensing a compound of formula (II)

wherein R¹ and R³ are as defined above and R^(2a) is lower alkoxy, witha compound of formula (III)

wherein R⁴, R⁵ and R⁶ are as defined above to produce a peptidederivative of formula (I-a) or a pharmaceutically acceptable saltthereof

wherein R¹, R^(2a), R³, R⁴, R⁵ and R⁶ are as defined above.

6. A process for preparing a peptide derivative of formula (I) or apharmaceutically acceptable salt thereof, comprising the following step(ii):

-   (ii) oxidizing a compound of formula (I-a) to produce a peptide    derivative of formula (I-b) or a pharmaceutically acceptable salt    thereof

wherein R¹, R^(2a), R³, R⁴, R⁵ and R⁶ are as defined above.

7. A process for preparing a peptide derivative of formula (I) or apharmaceutically acceptable salt thereof, comprising the following step(iii):

-   (iii) treating a compound of formula (I-a) or (I-b) with an acid to    produce a peptide derivative of formula (I-c) or a pharmaceutically    acceptable salt thereof

wherein X, R¹, R³, R⁴, R⁵ and R⁶ are as defined above.

8. An Arg-gingipain and Lys-gingipain inhibitor comprising as an activeingredient at least one member selected from the group consisting of apeptide derivative of formula (I) according to item 1 and apharmaceutically acceptable salt thereof.

9. A pharmaceutical preparation for periodontal disease comprising as anactive ingredient at least one member selected from the group consistingof a peptide derivative of formula (I) according to item 1 and apharmaceutically acceptable salt thereof.

10. A composition for use in the oral cavity comprising apharmaceutically acceptable carrier and at least one member selectedfrom the group consisting of a peptide derivative of formula (I)according to item 1 and a pharmaceutically acceptable salt thereof.

11. A method of preventing periodontal disease, comprising administeringan effective amount of the Arg-gingipain and Lys-gingipain inhibitor ofitem 8 to a mammal including a human.

12. A method of preventing periodontal disease, comprising administeringan effective amount of the pharmaceutical preparation for periodontaldisease of item 9 to a mammal including a human.

13. A method of preventing periodontal disease, comprising administeringan effective amount of the composition for use in the oral cavity ofitem 10 to a mammal including a human.

14. A method of treating periodontal disease, comprising administeringan effective amount of the Arg-gingipain and Lys-gingipain inhibitor ofitem 8 to a mammal including a human with periodontal disease.

15. A method of treating periodontal disease, comprising administeringan effective amount of the pharmaceutical preparation for periodontaldisease of item 9 to a mammal including a human with periodontaldisease.

16. A method of treating periodontal disease, comprising administeringan effective amount of the composition for use in the oral cavity ofitem 10 to a mammal including a human with periodontal disease.

17. Use of the peptide derivative of item 1 or a pharmaceuticallyacceptable salt thereof for preparing the Arg-gingipain andLys-gingipain inhibitor of item 8.

18. Use of the peptide derivative of item 1 or a pharmaceuticallyacceptable salt thereof for preparing the pharmaceutical preparation forperiodontal disease of item 9.

19. Use of the peptide derivative of item 1 or a pharmaceuticallyacceptable salt thereof for preparing the composition for use in theoral cavity of item 10.

In formula (I), the amino protecting group represented by R¹ is notspecifically limited as long as it does not adversely affect livingorganisms and synthetic reactions. Commonly used amino protectinggroups, such as those described in T. W. Greene, “Protective groups inOrganic Synthesis”, A Wiley-Interscience Publication, John-Wiley & Sons,New York, 1981, pp. 218–287, are suitable. Specific examples includeoptionally substituted aralkyloxycarbonyl, optionally substituted loweralkyloxycarbonyl, substituted sulfonyl, acetyl, benzyl,1-adamantyloxycarbonyl, cyclopentyloxycarbonyl and the like.

Examples of optionally substituted aralkyloxycarbonyl groups includebenzyloxycarbonyl (Cbz); benzyloxycarbonyl substituted with 1 to 3 C₁₋₄lower alkoxy groups, such as p-methoxybenzyloxycarbonyl andp-ethoxybenzyloxycarbonyl; benzyloxycarbonyl substituted with a nitrogroup, such as p-nitrobenzyloxycarbonyl; benzyloxycarbonyl substitutedwith 1 to 3 halogen atoms, such as p-bromobenzyloxycarbonyl and2,4-dichlorobenzyloxycarbonyl; diphenylmethoxycarbonyl and the like.

Examples of optionally substituted lower alkyloxycarbonyl groups includeC₂₋₇ straight or branched chain lower alkyloxycarbonyl optionallysubstituted with 1 to 3 halogen atoms, such as methoxycarbonyl,ethoxycarbonyl, t-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl and2,2,2-trichloroethyloxycarbonyl.

Examples of the substituted sulfonyl group include sulfonyl having onesubstituent such as a C₁₋₆ straight or branched chain lower alkyl groupor a phenyl group optionally substituted with 1 to 3 C₁₋₆ straight orbranched chain lower alkyl groups, such as benzenesulfonyl,p-toluenesulfonyl and methanesulfonyl.

Preferably, the amino protecting group represented by R¹ is anoptionally substituted aralkyloxycarbonyl group or an optionallysubstituted lower alkyloxycarbonyl group. Especially preferable isoptionally lower alkoxy-, nitro- or halogen-substitutedbenzyloxycarbonyl, or optionally halogen-substituted C₂₋₇ straight orbranched chain lower alkyloxycarbonyl. Particularly preferable isbenzyloxycarbonyl or 2,2,2-trichloroethyloxycarbonyl.

Examples of lower alkoxy groups represented by R² include C₁₋₆ straightor branched chain lower alkoxy groups such as methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, isobutoxy and t-butoxy. Preferable ist-butoxy.

The side chain (R group) of lysine represented by R³ or R⁴ means theside chain or residue bonded to the α-carbon atom of lysine, i.e.,4-aminobutyl. The amino group of the side chain (R group) may beprotected with a protecting group. Examples of useful amino protectinggroups are the above-mentioned amino protecting groups. Preferably, theamino protecting group is a C₂₋₇ straight or branched chain loweralkyloxycarbonyl group optionally substituted with 1 to 3 halogen atoms.Especially preferable are unsubstituted C₂₋₇ straight or branched chainlower alkyloxycarbonyl groups. Particularly preferable ist-butoxycarbonyl.

The side chain (R group) of arginine means the side chain or residuebonded to the α-carbon atom of arginine, i.e., 3-guanidinopropyl. Theguanidino group of the side chain (R group) may be protected with aprotecting group. The protecting group is not specifically limited aslong as it does not adversely affect living organisms and syntheticreactions. Commonly used guanidino protecting groups, such as thosedescribed in T. W. Greene, “Protective groups in Organic Synthesis”, AWiley-Interscience Publication, John-Wiley & Sons, New York, 1981, pp.218–287, are suitable for use. Specific examples include nitro; sulfonylsubstituted with one substituent such as phenyl optionally substitutedwith 1 to 3 C₁₋₆ straight or branched chain lower alkyl groups, orchromane optionally substituted with 1 to 6 C₁₋₆ straight or branchedchain lower alkyl groups, such as p-toluenesulfonyl and2,2,5,7,8-pentamethylchromane-6-sulfonyl; and oxycarbonyl groupssubstituted with one substituent such as aralkyl or adamantyl, such asbenzyloxycarbonyl, phenethyloxycarbonyl and 1-adamantyloxycarbonyl.Preferable is nitro.

Examples of lower alkyl groups represented by R⁵ and R⁶ are C₁₋₆straight or branched chain lower alkyl groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 1-ethylbutyl and the like.Preferable are methyl and ethyl.

Examples of aralkyl groups include phenyl-C₁–C₆ alkyl, particularlybenzyl and phenethyl.

Of the compounds of formula (I), preferable are those wherein X is —CO—.Especially preferable are those wherein X is —CO—, R¹ is hydrogen oroptionally substituted aralkyloxycarbonyl, R² is hydroxyl or C₁₋₆straight or branched chain lower alkoxy, one of R³ and R⁴ is the sidechain (R group) of lysine and the other of R³ and R⁴ is the side chain(R group) of arginine the guanidino group of which may be protected witha nitro group, and R⁵ and R⁶ may be the same or different and arehydrogen or aralkyl. Particularly preferable compounds are those whereinX is —CO—, R¹ is benzyloxycarbonyl, R² is hydroxyl or t-butoxy, R³ isthe side chain (R group) of lysine, R⁴ is the side chain (R group) ofarginine the guanidino group of which is protected with a nitro group,R⁵ is hydrogen and R⁶ is phenethyl.

The pharmaceutically acceptable salts of the compounds of the inventionare not particularly limited. Examples thereof include acid additionsalts formed by reacting the compounds of the invention withpharmaceutically acceptable acids. Specific examples include inorganicacid salts such as hydrochlorides and sulfates; and organic acid saltssuch as formates, trifluoroacetates, acetates, tartrates, maleates,fumarates, succinates and methanesulfonates. The compounds of theinvention or pharmaceutically acceptable salts thereof may be in theform of solvates such as hydrates.

The amino acids constituting the compound of the invention may be L- orD-amino acids. Preferable are L-amino acids.

The compounds of the invention may exist as enantiomers ordiastereoisomers depending on the asymmetric carbons in the molecularstructure. All such enantiomers and diastereoisomers are included in thescope of the invention. Such compounds can be used as is as isomericmixtures or can be optically resolved by conventional techniques.

The compounds of formula (I) can be prepared according to the followingreaction schemes.

wherein R¹, R³, R⁴, R⁵ and R⁶ are as defined above; and R^(2a) is loweralkoxy.

Examples of lower alkoxy groups represented by R^(2a) include C₁₋₆straight or branched chain lower alkoxy groups, preferably methoxy,ethoxy and t-butoxy.

Step (i): The compound of formula (I-a) of the invention can besynthesized by condensing a compound of formula (II) with a knowncompound of formula (III) prepared according to the method described inJapanese Unexamined Patent Publication No. 1999-228526 in a suitablesolvent.

The condensation reaction can be carried out by conventional methods.Useful methods include, for example, methods using a condensing agentsuch as N,N-dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or the like;methods using additives (e.g., 1-hydroxybenzotriazole,N-hydroxy-5-norbornene-2,3-dicarboxyimide, etc.) in addition to thecondensing agent; mixed acid anhydride methods using isobutylchloroformate, etc.; azide methods; active ester methods; and the like.

Any solvent can be used in the condensation reaction so long as it isinert to the reaction. Useful solvents include, for example,N,N-dimethylformamide, tetrahydrofuran, methylene chloride, dioxane,ethyl acetate, N-methylpyrrolidone and the like. These solvents can beused singly or in combination of two or more. The amount of compound offormula (III) is about 0.5 to about 10 moles, and preferably about 1 toabout 5 moles, per mole of the compound of formula (II). The amount ofcondensing agent is about 0.5 to about 10 moles, preferably about 1 toabout 5 moles, per mole of compound of formula (II). The reaction timeis about 0.3 to about 100 hours, and preferably about 0.5 to about 20hours. The reaction temperature is about −20° C. to about 100° C., andpreferably about 0° C. to about 40° C. The compound obtained in thisstep can be used in the following reaction step, with or without beingisolated.

wherein R¹, R^(2a), R³, R⁴, R⁵ and R⁶ are as defined above.

Step (ii): The compound of formula (I-b) of the invention can besynthesized by oxidizing the compound of formula (I-a) obtained inReaction Scheme 1 in a suitable solvent.

The oxidation reaction can be carried out by conventional methods.Useful methods include, for example, Dess-Martin oxidation usingDess-Martin reagents; improved Moffat oxidation using dimethylsulfoxide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride/dichloroacetic acid; and oxidation usingN-tert-butylphenylsulfinimidoyl chloride.

Any solvent can be used in the oxidation reaction so long as it is inertto the reaction. Useful solvents include, for example,N,N-dimethylformamide, tetrahydrofuran, methylene chloride, dioxane,ethyl acetate, acetonitrile, N-methylpyrrolidone and the like. Thesesolvents can be used singly or in combination of two or more. The amountof oxidizing reagent is about 0.3 to about 100 moles, preferably about 1to about 10 moles, per mole of compound of formula (I-a). The reactiontime is about 0.1 to about 100 hours, preferably about 0.5 to about 50hours. The reaction temperature is about −78° C. to about 100° C., andpreferably about 0° C. to about 40° C. The compound obtained in thisstep can be used in the following reaction step, with or without beingisolated.

wherein X, R¹, R³, R⁴, R⁵ and R⁶ are as defined above.

Step (iii): The compound of formula (I-c) of the invention can beobtained by treating a compound of formula (I-a) or (I-b) obtained inReaction Scheme 1 or 2 with an acid, in a suitable solvent or in theabsence of solvents.

Any solvent can be used in the reaction as long as it is inert to thereaction. Useful solvents include, for example, chloroform, methylenechloride, dioxane, tetrahydrofuran and ethyl acetate. Useful acidsinclude, for example, mineral acids such as hydrochloric acid andsulfuric acid; and organic acids such as trifluoroacetic acid andp-toluenesulfonic acid. The amount of acid used in the reaction is about1 to about 1,000 moles, and preferably about 1 to about 100 moles, permole of the compound of formula (I-a) or (I-b). The reaction time isabout 0.5 to about 50 hours. The reaction temperature is about 0° C. toabout 100° C., and preferably about 0° C. to about 30° C.

The compound of formula (II) can be produced, for example, according tothe following Reaction Scheme 4:

wherein R¹, R^(2a) and R³ are as defined above and R⁷ is lower alkoxy.

Examples of lower alkoxy groups represented by R⁷ include theabove-mentioned C₁₋₆ straight or branched chain lower alkoxy groups.Preferable are methoxy, ethoxy and t-butoxy.

Step (iv): The compound of formula (VI) of the invention can be producedby condensing a compound of formula (IV) with a known compound offormula (V) in a suitable solvent.

The condensation reaction can be carried out by conventional methods.Useful methods include, for example, methods using a condensing agentsuch as N,N-dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or the like;methods using additives (e.g., 1-hydroxybenzotriazole,N-hydroxy-5-norbornene-2,3-dicarboxyimide, etc.) in addition to thecondensing agent; mixed acid anhydride methods using isobutylchloroformate, etc.; azide methods; and active ester methods.

Any solvent can be used in the condensation reaction so long as it isinert to the reaction. Useful solvents include, for example,N,N-dimethylformamide, tetrahydrofuran, methylene chloride, dioxane,ethyl acetate, N-methylpyrrolidone and the like. These solvents can beused singly or in combinations of two or more. The amount of compound offormula (V) is about 0.5 to about 10 moles, preferably about 1 to about5 moles, per mole of compound of formula (IV). The amount of condensingagent is about 0.5 to about 10 moles, preferably about 1 to about 5moles, per mole of compound of formula (IV). The reaction time is about0.3 to about 100 hours, and preferably about 0.5 to about 20 hours. Thereaction temperature is about −10° C. to about 100° C., and preferablyabout 0° C. to about 40° C. The compound obtained in this step can beused in the following reaction step (v), with or without being isolated.

The compound of formula (IV) can be obtained by subjecting a knowncompound described in WIPO Publication No. WO 98/50420, JapaneseUnexamined Patent Publication No. 1999-228526, etc. to a conventionalamino group deprotection reaction. Useful deprotection reactionsinclude, for example, catalytic hydrogenation, methods usingtrimethylsilyliodide or triethylsilane, and the like. Catalysts usablein catalytic reduction methods include, for example, palladium-carbon,palladium chloride and the like. Since deprotection conditions varydepending on the type of protecting group of the precursor compound, thecompound of formula (IV) may be obtained in the free state or as a salt.Such salts are not particularly limited so long as they do notparticipate in the condensation reaction. Specific examples thereofinclude mineral acid salts such as hydrochlorides and sulfates; andorganic acid salts such as p-toluenesulfonates and methanesulfonates.

Step (v): The compound of formula (II) can be obtained by hydrolysis ofthe compound (VI) obtained in the above step (iv) with a base in asuitable solvent. Any solvent can be used in the reaction so long as itis inert to the reaction. For example, water, methanol, ethanol,1-propanol, 2-propanol, tetrahydrofuran and the like can be used singlyor in combination of two or more. Examples of useful bases includelithium hydroxide, sodium hydroxide, potassium hydroxide and the like.The amount of base is about 1 to about 10 moles, and preferably about 1to about 2 moles, per mole of the compound of formula (VI). The reactiontime is about 0.3 to about 100 hours, and preferably about 0.5 to about20 hours. The reaction temperature is about 0 to about 100° C., andpreferably about 0 to about 40° C. The compound obtained in this stepcan be used in Reaction Scheme 1, with or without being isolated.

The compounds of the invention obtained by the above processes and theother above-mentioned compounds can be purified by the separation andpurification techniques typically used in the field of chemicalsynthesis, such as recrystallization, distillation and various columnchromatographic techniques.

The peptide derivative of formula (I) of the invention andpharmaceutically acceptable salts thereof potently inhibit KGP and RGP,proteolytic enzymes produced by P. gingivalis closely associated withthe onset and progress of periodontal disease. The peptide derivative ofthe invention is composed of highly safe natural amino acids orderivatives thereof. Therefore, the peptide derivative and itsmetabolites produced in vivo are assumed to be highly safe.

Therefore, the peptide derivative of formula (I) and pharmaceuticallyacceptable salts thereof are useful as an active ingredient ofLys-gingipain and Arg-gingipain inhibitors and pharmaceuticalpreparations for periodontal disease. Such Lys-gingipain andArg-gingipain inhibitors and pharmaceutical preparations for periodontaldisease can be used as periodontal preventive agents and therapeuticagents.

The peptide derivative of formula (I) and pharmaceutically acceptablesalts thereof can also be used, together with a pharmaceuticallyacceptable carrier, to prepare compositions for use in the oral cavity.The peptide derivative of the invention or pharmaceutically acceptablesalts thereof can be mixed with a pharmaceutically acceptable carrierand administered as preparations for use in the oral cavity, such as gelpreparations for use in the oral cavity, oral ointments for adhesiveapplication to mucous membranes, oral pastes, periodontal-pocketintercalating agents, and preparations for adhesive application togingivae; and oral hygiene agents such as dentifrices, mouthwashes,chewing gums, tablets, candies, troches and the like. The compositionsfor oral cavity can be used as periodontal preventive agents ortherapeutic agents.

Useful pharmaceutically acceptable carriers include appropriate carrierscommonly used in accordance with the dosage form. Specific examplesinclude methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose,carboxymethylcellulose sodium, hydroxypropylmethylcellulose, liquidparaffin, white petrolatum, platinum base, Eudragit L, sodium alginate,propylene glycol alginate, pullulan, tragacanth, xanthan gum, chitosan,polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, sodiumpolyacrylate, polymethacrylic acid, ethyl methacrylate, dimethylaminoacetate, cellulose acetate, collagen, atherocollagen, gelatin, glycerol,triacetin, Macrogol 400, Polysorbate 60, polyoxyl stearate 40, butylp-hydroxybenzoate, ethanol, cetyl alcohol, glyceryl monostearate,calcium carbonate, magnesium carbonate, calcium secondary phosphate,carrageenan, sodium dioctyl sulfosuccinate, sodium lauryl sulfate,sodium dodecylbenzenesulfonate, hinokitiol, allantoin, glycyrrhizin, gumarabic, starch, cornstarch, saccharin, saccharin sodium, stevioside,glucose, lactose, sorbitol, mannitol, magnesium stearate, monobasicpotassium phosphate, dibasic potassium phosphate, menthol, eucalyptusoil, peppermint, spearmint, colors, aromas, sodium fluoride, sodiummonofluorophosphate and like fluorides, lysozyme chloride, azulene andlike anti-inflammatory agents, and sodium chloride and like typicallyadded components.

When the Lys-gingipain and Arg-gingipain inhibitor, pharmaceuticalpreparation for periodontal disease or composition for use in oralcavity, each containing the peptide derivative of the invention or apharmaceutically acceptable salt thereof as an active ingredient, isadministered to a mammal, including a human, usable administrationmethods are such that the preparation is inserted, applied or used forwashing in a suitable amount at least once a day with an activeingredient content of about 0.001 wt. % or more, and preferably about0.01 to about 20 wt. %.

When the Lys-gingipain and Arg-gingipain inhibitor, pharmaceuticalpreparation for periodontal disease or composition for use in the oralcavity of the invention is used as a therapeutic agent, the dosage canbe suitably selected according to the mode of administration, the age,gender and other conditions of the patient, and the severity of disease.When administered to a human, the dosage of the active ingredientcompound of the invention is usually about 0.001 to about 100 mg per kgbody weight per day, and preferably about 0.005 to about 10 mg/kg/day.

When the Lys-gingipain and Arg-gingipain inhibitor, pharmaceuticalpreparation for periodontal disease or composition for oral cavity ofthe invention is used as preventive agents, the dosage can be suitablyselected according to the mode of administration, the age, gender andother conditions of the human or other mammal. When administered to ahuman, the dosage of the active ingredient compound of the invention isusually about 0.001 to about 100 mg per kg body weight per day,preferably about 0.005 to about 10 mg/kg/day.

BEST MODE FOR CARRYING OUT THE INVENTION

The following Reference Examples, Examples, Formulation Examples andTest Examples are provided to illustrate the invention in more detail.It is to be understood that the invention is not limited to theExamples. In the examples, Me represents methyl; Boc, t-butoxycarbonyl;Cbz, benzyloxycarbonyl; t-Bu, t-butyl; and Ph, phenyl. Abbreviations foramino acids follow the generally used recommendations of the IUPAC-IUB.

REFERENCE EXAMPLE 1

The following compound, included in the compound of formula (VI) shownin Reaction Scheme 4, was synthesized.

Four grams of 10% palladium-carbon was added to 1,000 ml of a mixedmethanol/chloroform (10:1) solution containing 21 g (49.5 mmol) ofmethyl(3S)-3-benzyloxycarbonylamino-7-tert-butoxycarbonylamino-2-hydroxyheptanoate(Cbz-Lys(Boc)ψ[CHOHCO]-OMe), i.e., a known compound prepared accordingto the method described in WO 98/50420. The resulting mixture wasstirred under a hydrogen atmosphere at room temperature for 3 hours and45 minutes to eliminate the benzyloxycarbonyl protecting group from theamino group of lysine. After completion of the reaction, insolublematter was filtered off. The filtrate was concentrated and then, withoutisolation and purification, dissolved in 525 ml of N,N-dimethylformamide(DMF). Twenty grams (59.4 mmol) of N-benzyloxycarbonyl-L-glutamic acidγ-t-butylester (Cbz-Glu(O-t-Bu)-OH), 8.7 g (64.3 mmol) of1-hydroxybenzotriazole, 11.4 g (59.4 mmol) of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 13.5 g(134 mmol) of N-methylmorpholine were added to this solution withice-cooling, and the mixture was stirred at room temperature for 14hours. After completion of the reaction, a 10% aqueous citric acidsolution was added to adjust the pH to 3 and the reaction mixture wasextracted with ethyl acetate. The ethyl acetate layer was washedsequentially with a saturated saline solution, a 5% aqueous sodiumhydrogen carbonate solution and a saturated saline solution, and driedover anhydrous sodium sulfate. After filtration, the solvent wasdistilled off and the residue was separated and purified by silica gelcolumn chromatography (hexane:ethyl acetate=3:2 to 1:1), giving 18.3 gof a diastereomeric mixture of the desired compound as a white powder(yield: 60%). The following are the physical properties of the mixture.

¹H-NMR(DMSO-d₆)δ: 7.67 (0.3H, d, J=8.8 Hz), 7.54 (0.7H, d, J=9.0 Hz),7.40–7.25 (6H, m), 6.73 (1H, m), 5.69 (0.3H, d, J=5.9 Hz), 5.53 (0.7H,d, J=5.6 Hz), 5.02 (2H, m), 4.11–3.96 (3H, m), 3.61 & 3.56 (3H, s), 2.87(2H, m), 2.20 (2H, m), 1.83 (1H, m), 1.66 (1H, m), 1.59–1.06 (6H, m),1.38 (9H, s), 1.36 (9H, s)

Mass (FAB(+)): 610 (M+H)⁺

State: white powder

m.p.: 101 to 103° C.

REFERENCE EXAMPLE 2

The following compound, included in the compound of formula (II) shownin Reaction Scheme 4, was synthesized.

An aqueous solution (10 ml) of 270 mg (6.43 mmol) of lithium hydroxidemonohydrate was added to 100 ml of a tetrahydrofuran (THF) solution of3.53 g (5.76 mmol) of the compound obtained in Reference Example 1 withice cooling. The resulting mixture was stirred with ice-cooling for 1hour and at room temperature for 30 minutes. After completion of thereaction, the reaction mixture was concentrated under reduced pressure.A 10% aqueous solution of citric acid was added to the residue to adjustthe pH to 3, followed by extraction with ethyl acetate. The ethylacetate layer was washed with a saturated saline solution and then driedover anhydrous sodium sulfate. After filtration, the solvent wasdistilled off, giving 3.5 g of the desired compound as a white powder(yield: 100%). The following are the physical properties of thecompound.

¹H-NMR(DMSO-d₆)δ: 12.45 (1H, br s), 7.49–7.31 (7H, m), 6.74 (1H, m),5.38 (0.3H, d, J=5.8 Hz), 5.26 (0.7H, d, J=5.8 Hz), 5.01 (1H, q, J=12.4Hz), 4.04–3.85 (4H, m), 2.86 (2H, m), 2.19 (2H, m), 1.90–0.90 (8H, m),1.40–1.36 (18H, s×2)

Mass (FAB(−)): 596 (M+H)⁺, 594 (M−H)⁻

State: white powder

m.p.: 48 to 50° C.

EXAMPLE 1

The following compound of the invention was synthesized.

One hundred and seventy two milligrams (0.44 mmol) of hydrochloride saltof 6-nitroguanidino-(3S)-amino-2-hydroxyheptanoic acid N-phenethylamide(H-Arg(N^(g)NO₂)—CH(OH)—CONHCH₂CH₂Ph) described in Japanese UnexaminedPatent Publication No. 1996-502493, 63 mg (0.46 mmol) of1-hydroxybenzotriazole, 89 mg (0.46 mmol) of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 89 mg(0.88 mmol) of N-methylmorpholine were added to 4 ml of a DMF solutionof 263 mg (0.44 mmol) of the compound obtained in Reference Example 2with ice-cooling, and the resulting mixture was stirred at roomtemperature for 3.5 hours. After completion of the reaction, a 10%aqueous citric acid solution was added to adjust the pH to 3, followedby extraction with ethyl acetate. The ethyl acetate layer was washedsequentially with a saturated saline solution, a 5% aqueous sodiumhydrogen carbonate solution and a saturated saline solution, and driedover anhydrous sodium sulfate. After filtration, the solvent wasdistilled off and the residue was separated and purified by silica gelcolumn chromatography (chloroform:methanol=20:1), giving 190 mg of adiastereomeric mixture of the desired compound as an amorphous solid(yield: 46%). The following are the physical properties of the mixture.

¹H-NMR(DMSO-d₆)δ: 8.50 (0.5H, m), 8.25–7.17 (14.5H, m), 6.75–6.58(1H,m), 6.01–5.75 (2H, m), 4.99 (2H, m), 4.25–3.82 (5H, m), 3.48–2.97(4H,m), 2.91–2.65 (4H, m), 2.20 (2H, m), 1.95–1.58 (2H, m), 1.52–1.11 (28H,m)

Mass(FAB(+)); 952 (M+Na)⁺, 968 (M+K)⁺

State: amorphous

EXAMPLE 2

The following compound of the invention was synthesized.

One hundred and forty four milligrams (0.34 mmol) of Dess-Martin reagentwas added to 2 ml of a methylene chloride solution of 105 mg (0.11 mmol)of the compound obtained in Example 1, and the mixture was stirred atroom temperature for 1 hour. After completion of the reaction, a 20%aqueous sodium hydrogen sulfite solution was added. The mixture wasstirred for 5 minutes and extracted with ethyl acetate. The ethylacetate layer was washed sequentially with a saturated saline solution,a 5% aqueous sodium hydrogen carbonate solution and a saturated salinesolution, and then dried over anhydrous sodium sulfate. Afterfiltration, the solvent was distilled off, and the residue was separatedand purified by silica gel column chromatography(chloroform:methanol=40:1), giving 101 mg of the desired compound as anamorphous solid (yield: 97%). The following are the physical propertiesof the compound.

¹H-NMR(DMSO-d₆)δ: 9.10–8.00 (3H, m), 7.55–7.15 (12H, m), 6.80–6.62 (1H,m), 5.06–4.94 (3H, m), 4.18–3.91 (2H, m), 3.21–2.65 (8H, m), 2.29–2.18(2H, m), 1.86–0.98 (30H, m)

LC/Mass ES(+); 925 (M⁺), ES(−); 924 (M−H)⁻

State: amorphous

EXAMPLE 3

The following compound of the invention was synthesized.

Two milliliters of a 4N hydrochloric acid-ethyl acetate solution wasadded to 75 mg (0.08 mmol) of the compound obtained in Example 2, andthe mixture was stirred at room temperature for 40 minutes. Anhydrousether was added to the reaction mixture and the resulting whiteprecipitate was collected by filtration and washed with anhydrous ether,giving 59 mg of the desired compound as an amorphous solid (yield: 90%).The following are the physical properties of the compound.

¹H-NMR(DMSO-d₆)δ: 9.10–8.30 (3H, m), 7.75 (3H, br s), 7.60–7.11 (12H,m), 6.80–6.60 (1H, m), 5.06–4.95 (3H, m), 4.20–3.95 (2H, m), 3.20–2.61(8H, m), 2.31–2.18 (2H, m), 1.86–1.00 (12H, m)

Mass(FAB(+)); 770 (M+H)⁺, 768 (M−H)⁻

State: amorphous

Formulation Example 1 Oral ointment Compound 1 of the invention  1.0obtained in Example 1 White petrolatum  10.0 Sodium polyacrylate  3.0Liquid paraffin Balance Total 100.0 (wt. %)

An oral ointment was prepared in a conventional manner according to theabove formulation.

Formulation Example 2 Dentifrice Calcium secondary phosphate  42.0Glycerin  19.0 Carrageenan  0.9 Sodium lauryl sulfate  1.2 Saccharin 1.0 Compound of the invention  1.0 obtained in Example 2 Butylp-hydroxybenzoate  0.005 Flavor  1.0 Water Balance Total 100.0 (wt. %)

A dentifrice was prepared in a conventional manner according to theabove formulation.

Formulation Example 3 Troche Gum arabic  6.0 Glucose  72.0 Lactose  19.0Compound of the invention  1.5 obtained in Example 3 Sodiummonofluorophosphate  0.7 Flavor  1.0 Water Balance Total 100.0 (wt. %)

Troches were prepared in a conventional manner according to the aboveformulation.

Formulation Example 4 Chewing gum Polyvinyl acetate  20.0Polyisobutylene  3.0 Calcium carbonate  2.0 Sorbitol  55.0 Mannitol 15.0 Compound of the invention  4.0 obtained in Example 3 Flavor  1.0Total 100.0 (wt. %)

Chewing gum was prepared in a conventional manner according to the aboveformulation.

Formulation Example 5 Gargle Ethanol  20.0 Polyoxyethylene (60)  3.0hydrogenated castor oil Polyethylene glycol  2.0 Glycerin  10.0 Sodiumsaccharin  0.02 Compound of the invention  0.5 obtained in Example 3Flavor  0.2 Water Balance Total 100.0 (wt. %)

A gargle was prepared in a conventional manner according to the aboveformulation.

Formulation Example 6 Mouthwash Ethanol  30.0 Polyoxyethylene (20)  1.0sorbitan laurate Polyoxyethylene (40)  0.5 hydrogenated castor oilSodium hydroxide  0.05 Sodium saccharin  0.05 Compound of the invention 0.5 obtained in Example 3 Flavor  0.5 Water Balance Total 100.0 (wt. %)

A mouthwash was prepared in a conventional manner according to the aboveformulation.

TEST EXAMPLE 1 Determination of Inhibitory Activities Against KGP andRGP

The inhibitory activity against Lys-gingipain (KGP) was measured by themethod of Abe et al. (Journal Biochemistry, 1998, Vol. 123, 305–312)using Cbz-His-Glu-Lys-MCA as a substrate. The inhibitory activityagainst Arg-gingipain (RGP) was measured by the method of Kadowaki etal. (Journal Biological Chemistry, 1994, Vol. 269, 21371–21378) usingCbz-Phe-Arg-MCA as a substrate. More specifically, the measurements werecarried out in the following manner: 100 μl of 50 mM L-cystein, 200 μlof a 0.1 M sodium phosphate buffer solution (pH 7.5), 20 μl of 12.3 nMKGP or RGP solution containing 0.05% “Brij35” (a tradename, product ofAldrich, polyoxyethylene(23) lauryl ether), 80 μl of distilled water and100 μl of a dimethyl sulfoxide solution of a compound according to theinvention were mixed and preincubated at 37° C. for 5 minutes.Thereafter, 500 μl of a 0.1% dimethyl sulfoxide solution containing 20μM Cbz-His-Glu-Lys-MCA (for KGP) or Cbz-Phe-Arg-MCA (for RGP) was added,followed by incubation at 40° C. for 10 minutes. An acetic acid buffersolution (pH 5.0) containing 10 mM iodoacetamide was then added to stopthe enzyme reaction. The fluorescence intensity (F) at 460 nm uponexcitation at 380 nm was measured. As a control, 100 μl of dimethylsulfoxide that did not contain a compound according to the invention wasused in place of a solution of the compound, and the fluorescenceintensity (F₀) was measured in a manner similar to the above. The enzymeinhibitory activity was calculated by the following equation:Enzyme inhibitory activity (%)=[1−(F/F ₀)]×100Table 1 shows the test results.

TABLE 1 Enzyme inhibitory Compound Concentration activity (%) (ExampleNo.) (mol/l) KGP RGP 3 10⁻⁴ 100.0 99.4 10⁻⁵ 99.9 96.8 10⁻⁶ 99.9 81.1

Table 1 shows that the compound of the invention has excellentinhibitory activities against both enzymes, KGP and RGP.

The compound of the invention inhibits both Lys-gingipain (KGP) andArg-gingipain (RGP) produced by a gram-negative anaerobic bacterium,Porphyromonas gingivalis, and is therefore useful, for example, as apreventive or therapeutic agent for periodontal disease.

1. A peptide derivative of formula (I) or a pharmaceutically acceptablesalt thereof

wherein X is —CH(OH)— or —CO—; R¹ is hydrogen or an amino protectinggroup; R² is hydroxyl or lower alkoxy; one of R³ and R⁴ is the sidechain (R group) of lysine the amino group of which may be protected witha protecting group and the other of R³ and R⁴ is the side chain (Rgroup) of arginine the guanidino group of which may be protected with aprotecting group; and R⁵ and R⁶ may be the same or different and arehydrogen, lower alkyl or aralkyl.
 2. The peptide derivative of formula(I) or a pharmaceutically acceptable salt thereof according to claim 1wherein X is —CO—.
 3. The peptide derivative of formula (I) or apharmaceutically acceptable salt thereof according to claim 2 wherein Xis —CO—; R¹ is hydrogen or optionally substituted aralkyloxycarbonyl; R²is hydroxyl or lower alkoxy; one of R³ and R⁴ is the side chain (Rgroup) of lysine and the other of R³ and R⁴ is the side chain (R group)of arginine the guanidino group of which may be protected with a nitrogroup; and R⁵ and R⁶ may be the same or different and are hydrogen oraralkyl.
 4. The peptide derivative of formula (I) or a pharmaceuticallyacceptable salt thereof according to claim 3 wherein X is —CO—; R¹ isbenzyloxycarbonyl; R² is hydroxyl or t-butoxy; R³ is the side chain (Rgroup) of lysine; R⁴ is the side chain (R group) of arginine theguanidino group of which is protected with a nitro group; R⁵ ishydrogen; and R⁶ is phenethyl.
 5. A process for preparing a peptidederivative of formula (I) or a pharmaceutically acceptable salt thereof

wherein X is —CH(OH)— or —CO—; R¹ is hydrogen or an amino protectinggroup; R² is hydroxyl or lower alkoxy; one of R³ and R⁴ is the sidechain (R group) of lysine the amino group of which may be protected witha protecting group and the other of R³ and R⁴ is the side chain (Rgroup) of arginine the guanidino group of which may be protected with aprotecting group; and R⁵ and R⁶ may be the same or different and arehydrogen, lower alkyl or aralkyl, the process comprising the followingstep (i): (i) condensing a compound of formula (II)

wherein R¹ and R³ are as defined above and R^(2a) is lower alkoxy, witha compound of formula (III)

wherein R⁴, R⁵, and R⁶ are as defined above to produce a peptidederivative of formula (I-a) or a pharmacuetically acceptable saltthereof

wherein R¹, R^(2a), R³, R⁴, R⁵ and R⁶ are as defined above.
 6. A processaccording to claim 5 for preparing a peptide derivative of formula (I)or a pharmaceutically acceptable salt thereof, comprising the followingstep (ii): (ii) oxidizing a compound of formula (I-a) to produce apeptide derivative of formula (I-b) or a pharmaceutically acceptablesalt thereof

wherein R¹, R^(2a), R³, R⁴, R⁵ and R⁶ are as defined above.
 7. A processaccording to claim 5 for preparing a peptide derivative of formula (I)or a pharmaceutically acceptable salt thereof, comprising the followingstep (iii): (iii) treating a compound of formula (I-a) with an acid toproduce a peptide derivative of formula (I-c) or a pharmaceuticallyacceptable salt thereof

wherein R¹, R², R³, R⁴, R⁵ and R⁶ are as defined above, and X is—CH(OH)—.
 8. A pharmaceutical preparation for periodontal diseasecomprising as an active ingredient at least one member selected from thegroup consisting of a peptide derivative of formula (I) according toclaim 1 and a pharmaceutically acceptable salt thereof.
 9. A compositionfor use in the oral cavity comprising a pharmaceutically acceptablecarrier and at least one member selected from the group consisting of apeptide derivative of formula (I) according to claim 1 and apharmaceutically acceptable salt thereof.
 10. A method of preventingperiodontal disease, comprising administering an effective amount of thepharmaceutical preparation for periodontal disease of claim 8 to amammal including a human.
 11. A method of preventing periodontaldisease, comprising administering an effective amount of the compositionfor use in the oral cavity of claim 9 to a mammal including a human. 12.A method of treating periodontal disease, comprising administering aneffective amount of the pharmaceutical preparation for periodontaldisease of claim 8 to a mammal including a human with periodontaldisease.
 13. A method of treating periodontal disease, comprisingadministering an effective amount of the composition for use in the oralcavity of claim 9 to a mammal including a human with periodontaldisease.
 14. A process according to claim 6 for preparing a peptidederivative of formula (I) or a pharmaceutically acceptable salt thereof,comprising the following step (iii): (iii) treating a compound offormula (I-b) with an acid to produce a peptide derivative of formula(I-c) or a pharmaceutically acceptable salt thereof

wherein R¹, R³, R⁴, R⁵ and R⁶ are as defined above, and X is —CO—.